2-ethyl-3-methylpyrazine

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

24 February 2017 - 19 June 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

2-ethyl-3-methylpyrazine

EC Number:

239-799-8

EC Name:

2-ethyl-3-methylpyrazine

Cas Number:

15707-23-0

Molecular formula:

C7H10N2

IUPAC Name:

2-ethyl-3-methylpyrazine

Method

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9, induced by phenobarbitone/p-naphthoflavone

Test concentrations with justification for top dose:

Pre-Experiment/Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate
Experiment II: 33; 100; 333; 1000; 2500; and 5000 μg/plate

Vehicle / solvent:

- Vehicle/solvent used: DMSO

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 4-nitro-o-phenylene-diamine / 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: plate incorporation test (experiment I), pre-incubation test (experiment II)

DURATION
- Preincubation period: 60 minutes
- Exposure duration: ≥ 48 hours

NUMBER OF REPLICATIONS: 3 (two independent experiments)

DETERMINATION OF CYTOTOXICITY
- Method: counting numbers of revertants

Evaluation criteria:

A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed.
A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.

Statistics:

no statistical analysis

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at a concentration of 5000 µg/plate, induction factor < 0.5

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at a concentration of 5000 µg/plate, induction factor < 0.5

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No test material precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.

Any other information on results incl. tables

Table 1: Summary of Experiment I

Metabolic Activation	Test Group	Dose Level (per plate)	Revertant Colony Counts (Mean ± SD)
			TA 1535	TA 1537	TA 98	TA 100	WP2 uvrA
Without Activation	DMSO		13 ± 1	12 ± 3	28 ± 7	201 ± 37	41 ± 5
	Untreated		8±2	9±3	34±7	211±6	32±10
	Test Item	3 µg	12± 2	12±3	28±4	198±17	41±10
		10 µg	12±4	11±1	35±3	203±16	43±7
		33 µg	10±3	11±2	29±8	196±18	39±7
		100 µg	13±3	12±1	30±4	211±11	40±3
		333 µg	15±3	11±3	26±5	217±18	39±6
		1000 µg	11±1	10±1	26±1	214±20	33±9
		2500 µg	9±4	8±1	26±6	200±22	31±5
		5000 µg	12±2	5±1	24±3	151±18	24±6
	NaN3	10 µg	1467±38			2217±69
	4-NOPD	10 µg			564±50
	4-NOPD	50 µg		118±21
	MMS	2.0 µL					932± 18
With Activation	DMSO		14±5	13±4	34±3	162±22	50±9
	Untreated		12±3	26±7	43±6	211±24	51±2
	Test Item	3 µg	13±3	16±5	41±3	161±13	57±9
		10 µg	14±3	13±3	39±2	151±5	49±4
		33 µg	14±3	16±3	39±8	166±8	42±4
		100 µg	13±2	17±2	37±6	185±23	55±1
		333 µg	13±4	15±5	46±8	152±9	46±8
		1000 µg	9±2	18±4	36±4	162±10	46±10
		2500 µg	13±5	11±2	42±1	196±19	35±7
		5000 µg	13±3	13±2	33±4	189±7	33±10
	2-AA	2.5 µg	396±6	205±4	4701±749	3889±254
	2-AA	10.0 µg					463±19

NaN3: sodium azide, 2-AA: 2-aminoanthracene, 4 -NOPD: 4-nitro-o-phenylene-diamine, MMS: methyl methane sulfonate

Table 2: Summary of Experiment II

MetabolicActivation	Test Group	Dose Level (per plate)	Revertant Colony Counts (Mean ± SD)
			TA 1535	TA 1537	TA 98	TA 100	WP2 uvrA
Without Activation	DMSO		10 ± 3	9 ± 2	26 ± 7	148 ± 19	34 ± 5
	Untreated		10± 4	10± 5	31±7	206± 7	40± 8
	Test Item	33 µg	10± 4	9± 2	25± 8	134± 4	39± 6
		100 µg	9± 3	7± 2	26± 6	140± 28	33± 5
		333 µg	10± 4	7± 0	22± 3	115±18	39± 5
		1000 µg	9± 2	8± 1	22± 3	120± 9	35± 3
		2500 µg	9±2	5±1	22± 7	90± 5	24± 4
		5000 µg	8± 3	7± 3	21± 5	63± 4	26± 8
	NaN3	10 µg	1121± 154			1888± 171
	4-NOPD	10 µg			284± 17
	4-NOPD	50 µg		74± 5
	MMS	2.0 µL					547± 13
With Activation	DMSO		12± 2	14± 5	35± 2	120± 9	45± 8
	Untreated		14± 3	12± 4	40± 9	143± 36	51±6
	Test Item	33 µg	13±3	20± 3	39± 6	104± 10	43± 9
		100 µg	15±3	12± 4	35± 10	121± 26	42± 5
		333 µg	12± 2	15± 6	25± 6	118± 9	44± 10
		1000 µg	14± 2	15± 4	42± 9	92±23	41± 7
		2500 µg	11± 1	17± 3	34± 8	119±17	27± 9
		5000 µg	12± 3	12± 4	37± 1	79± 4	29± 7
	2-AA	2.5 µg	343± 12	155± 7	4001± 547	2322± 303
	2-AA	10.0 µg					392±38

NaN3:sodium azide,2-AA: 2-aminoanthracene,4 -NOPD: 4-nitro-o-phenylene-diamine,MMS: methyl methane sulfonate

Table 3: Historical Data

Strain		Without S9 mix				With S9 mix
		Mean	SD	Min	Max	Mean	SD	Min	Max
TA 1535	Solvent control	12	2.5	6	25	12	2.5	7	26
	Untreated control	12	3.1	6	28	12	2.9	7	26
	Positive control	1130	143.1	334	1816	388	58.2	176	668
TA 1537	Solvent control	10	2.2	6	19	13	3.5	7	30
	Untreated control	11	2.7	5	21	14	4.0	7	31
	Positive control	82	12.7	43	157	191	60.8	83	434
TA 98	Solvent control	25	4.4	13	43	34	6.2	15	58
	Untreated control	27	4.9	12	43	37	6.5	11	57
	Positive control	378	73.7	211	627	3949	771.8	360	6586
TA 100	Solvent control	156	26.0	78	209	148	32.3	73	208
	Untreated control	176	23.6	79	217	172	25.4	85	218
	Positive control	1966	293.2	498	2767	3798	830.4	536	6076
WP2uvrA	Solvent control	41	5.6	27	63	50	6.8	28	72
	Untreated control	42	5.8	30	63	52	6.8	36	88
	Positive control	798	362.7	319	4732	378	112.6	167	1265

Mean = mean value of revertants/plate, SD = standard deviation, Min = minimal value, Max = maximal value

Applicant's summary and conclusion

Conclusions:

During the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.
Therefore, the test item is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.

Executive summary:

A bacterial reverse mutation assay (OECD 471) was performed to investigate the potential of the test item to induce gene mutations according to the plate incorporation test (experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate) and the pre-incubation test (experiment II: 33; 100; 333; 1000; 2500; and 5000 μg/plate) using the Salmonella typhimuriumstrains TA 1535, TA 1537, TA 98, TA 100, and the Escherichia colistrain WP2uvrA.

The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. No precipitation of the test item occurred up to the highest investigated dose. The plates incubated with the test item showed normal background growth up to 5000 μg/plate with and without S9 mix in all strains used.

Only slight toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in strain TA1537 and TA100.

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with the test item at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

The controls confirmed the validity of the study.